ABCB4 p.Glu1125Gln
| Predicted by SNAP2: | A: D (85%), C: D (85%), D: D (80%), F: D (91%), G: D (91%), H: D (85%), I: D (91%), K: D (91%), L: D (91%), M: D (85%), N: D (91%), P: D (95%), Q: D (80%), R: D (91%), S: D (91%), T: D (91%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Mutational analysis of conserved carboxylate resid... Biochemistry. 2000 Nov 21;39(46):14138-49. Urbatsch IL, Julien M, Carrier I, Rousseau ME, Cayrol R, Gros P
Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein.
Biochemistry. 2000 Nov 21;39(46):14138-49., [PMID:11087362]
Abstract [show]
Mutagenesis was used to investigate the functional role of six pairs of aspartate and glutamate residues (D450/D1093, E482/E1125, E552/E1197, D558/D1203, D592/D1237, and E604/E1249) that are highly conserved in the nucleotide binding sites of P-glycoprotein (Mdr3) and of other ABC transporters. Removal of the charge in E552Q/E1197Q and D558N/D1203N produced proteins with severely impaired biological activity when the proteins were analyzed in yeast cells for cellular resistance to FK506 and restoration of mating in a ste6Delta mutant. Mutations at other acidic residues had no apparent effect in the same assays. These four mutants were expressed in Pichia pastoris, purified to homogeneity, and biochemically characterized with respect to ATPase activity. Studies with purified proteins showed that mutants D558N and D1203N retained 14 and 30% of the drug-stimulated ATPase activity of wild-type (WT) Mdr3, respectively, and vanadate trapping of 8-azido[alpha-(32)P]nucleotide confirmed slower basal and drug-stimulated 8-azido-ATP hydrolysis compared to that for WT Mdr3. The E552Q and E1197Q mutants showed no drug-stimulated ATPase activity. Surprisingly, drugs did stimulate vanadate trapping of 8-azido[alpha-(32)P]nucleotide in E552Q and E1197Q at a level similar to that of WT Mdr3. This suggests that formation of the catalytic transition state can occur in these mutants, and that the bond between the beta- and gamma-phosphates is hydrolyzed. In addition, photolabeling by 8-azido[alpha-(32)P]nucleotide in the presence or absence of drug was also detected in the absence of vanadate in these mutants. These results suggest that steps after the transition state, possibly involved in release of MgADP, are severely impaired in these mutant enzymes.
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No. Sentence Comment
69 Mutagenic oligos for the D1093N, E1125Q, D1203, and E1249Q mutations were 5'-CTTTGCCATTTA- GAAACAC-3', 5'-GGCAATGTTCTGCGCAATGCTGCAG- TC-3', 5'-TCAGCTCTGAATACAGAAAG-3', and 5'-AAG- GTCAAGCAGCACGGC-3', respectively.
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ABCB4 p.Glu1125Gln 11087362:69:33
status: NEW137 Single-point mutations D450N, E482Q, E552Q, D558N, D592N, and E604Q were introduced into the NB1 of Mdr3, and mutations D1093N, E1125Q, E1197Q, D1203N, D1237N, and E1249Q were introduced independently into the NB2.
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ABCB4 p.Glu1125Gln 11087362:137:128
status: NEW153 Cells expressing WT Mdr3 or the NB1 mutants D450N, E482Q, D592N, and E604Q (Figure 3A) or their NB2 counterparts D1093N, E1125Q, D1237N, and E1249Q (Figure 3B) were all resistant to FK506.
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ABCB4 p.Glu1125Gln 11087362:153:121
status: NEW169 Mating frequencies of mutants D450N (104%), E482Q (127%), D592N (22%), and E604Q (85%) in NB1 (Figure 4) or their counterparts D1093N (72%), E1125Q (101%), D1237N (68%), and E1249Q (118%) in NB2 (Figure 4) were similar to that of the Mdr3 WT control.
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ABCB4 p.Glu1125Gln 11087362:169:141
status: NEW259 Single-point mutations D450N, E482Q, D592N, and E604Q were introduced in NB1 and their homologous substitutions D1093N, E1125Q, D1237N, and E1249Q created in NB2, and the mutants were transformed in the yeast S. cereVisiae to assess their biological activity.
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ABCB4 p.Glu1125Gln 11087362:259:120
status: NEW